It has long been realized that there are distinct marketing and manufacturing advantages if one had the ability to control sex of the offspring in birds. Hens have the advantage over roosters for laying eggs. Roosters have the advantage over hens for meat production. Currently there is a 50% chance of obtaining the desired sex. For birds the sex of the offspring is determined by the female, male sperm have a homogenous sex chromosome content. This is in sharp contrast to mammals where sperm has a heterogenous sex chromosome content, bearing either the X-chromosome or the Y-chromosome. Consequently, avian females have haploid chromosomal diversity in the eggs. It has been demonstrated in Drosophila that there is a sex-specific initiation and maintenance of yolk protein synthesis, which may be conserved in chickens (Belote et al., 1985). Much of the egg yolk is synthesized by the adult female liver then added to the egg. Little is know about the post-translational changes that take place in yolk proteins as regulated by the haploid egg. Nothing is known about sex-specific protein contributions by the haploid cells that are a part of the egg or the early embryonic cell contributions. No one until now has identified those protein constituents. Bhattacharya et al. (U.S. Pat. No. 3,692,897) refers to an immunological method for controlling the sex of mammalian offspring, making use of spermatozoa which has been previously separated into fractions having the desired sex characteristics. He defines the sex as sperm that are genotypically known to contain X chromosomes which carry female producing genes, while others contain Y chromosomes which carry male producing genes. The claims and invention addresses antibodies reactive to X-sperm or Y-sperm from mammalian ejaculates. He does not anticipate or make obvious claims associated with avian germ cells nor does his invention refer to modifying eggs. Said antigens were derived from sperm and subsequent immunizations were to sperm immunogens. A method for isolating egg protein fractions or egg protein fractions from karyotyped avian eggs was not taught. The techniques herein described are available commercially, described in Gel Electrophoresis of Proteins: A Practical Approach and/or described in U.S. Pat. No. 5,021,243 to Spaulding.
Weak sex selection pressures will translate into large yields because poultry production is so large. There are several billion chickens produced every year. If sex selection pressures were weak, for example only affecting 1 birth in a 1000, those weak pressures would still improve the sex selection outcome by several million chickens per year. Therefore, weak selection pressures are amplified because the production numbers are so large. A novel approach for increasing the probability that a hen will have a specific sex offspring is described herein. The approach identifies embryonic sex-specific proteins. Those proteins are used in a vaccine for the hen. A vaccinated hen will transfer antibodies to the egg. Antibodies to sex-specific proteins in the egg will impede embryonic development thereby changing the sex selection pressures. Or, antibodies to sex-specific protein or sex-specific proteins could be used diagnostically. An approach for improving sex selection pressures similar to this approach has been successful in mammals (Calder and Rajamahendra, 1993). Their approach used mammalian spleen H-Y autoantigens in the vaccine.
Sex preselection pressures can be imposed through different approaches. One approach would be to vaccinate a hen with a sex-specific protein or a plurality of proteins. Antibodies, to sex-specific proteins, would transfer to the egg. As the embryo develops, it produces a new set of proteins that are distinct from the maternal set. Those sex-specific proteins are disclosed herein. Antibodies to evolving sex-specific proteins would impede embryogenesis by antibody sequestration thereby adding a selection pressure. Alternatively, testing the egg yolk for the presents of sex-specific proteins would provide information to decide which egg should be placed into an incubator. If females were the preferred sex, the yolk could be assayed for female sex-specific protein patterns using gel electrophoresis. Or, antibodies raised to sex-specific proteins could be used in an immunodetection assay. Either test would inform the producer about the sex of the egg.
A female ovum determines the sex of the offspring (Mizuno et. al., 1993), which is the opposite for mammals (U.S. Pat. No. 3,692,897; U.S. Pat. No. 5,021,244). Yolk proteins are synthesized by the maternal liver. Others yolk constituents are transferred from the maternal plasma. Therefore, it does not matter whether the egg is female or male they both start out with the same maternal complement of protein (Deeley et. al., 1993). A electrophoretic protein profile of a male or female egg, before embryonic development, would appear similar (a similar developmental identification system has been applied to identifying sex-specific proteins in mammalian sperm U.S. Pat. No. 5,080,895). The electrophoretic profiles would appear similar because the egg yolk proteins came from the same mother. Eggs begin with similar protein constituents. Later, as the embryo develops, unique stage specific proteins are expressed. Unique proteins would appear as unique spots or lines on a protein electrophoretogram. The proteins which are not unique, would appear as common spots seen on both female and male electrophoretograms (U.S. Pat. No. 5,080,895). Those unique proteins are associated with enabling development of organ systems. Sex-specific characteristics are seen early in the embryo (Deeley et. al., 1993; Mizuno et. al., 1993). Chromosomes for sex differentiation, expressed in stages during embryonic development, can be male or female specific, and may not be expressed in the adult (Deeley et. al., 1993; Mizuno et. al., 1993). The protein products of sex determination in poultry have not been reported, only the DNA specific expression has been reported (Mizuno et. al., 1993). It is known by those skilled in the art that there many proteins expressed during embryogenesis. Many of those proteins that are expressed during embryogenesis are unique to the embryo and not expressed in the adult. Each protein is vital. Progression from one stage to the next requires the orchestration of a complex set of proteins and cellular events (Eyal-Giladi, 1993). A loss or diminution of one protein type can have fatal consequences. Diminution may take the form of a single amino acid substitution that reduces the potency or the decreased production of a particular protein. The literature is replete with, and those skilled in the art are cognizant of, many diseases where reduced availability of a single protein has catastrophic consequences. It is also known that embryos are the most fragile and susceptible to aberrancies. Antibodies to sex-specific proteins would impose a selection pressure during a fragile period (Calder and Rajamahendra. 1993); a slight selection pressure would be amplified by the amount of poultry produced per year. Immunization methods for poultry are well known by those skilled in the art (Tsang and Grunder, 1983; Zijpp et. al., 1985; Gassmann et. al., 1990; U.S. Pat. No. 5,021,244). Maternal antibodies accumulate in the egg yolk in higher concentrations than maternal serum, and because of that characteristic, investigators have proposed eggs as a source for industrial production of antibodies (Fichtali et. al., 1993). There are two other unique features of poultry vaccination. Chickens have the potential to mount an antibody response to conserved mammalian protein (Gassmann et. al., 1990). Moreover, a hen can be vaccinated with an autoantigen (estradiol) and the antibodies will interfere with egg shell production (Tsang and Grunder, 1983). The route of antigen administration does not appear to affect the immune response (Zijpp et. al., 1985). Thus, hens have been vaccinated to autoantigens; the immune response has been shown to inhibit a growth function and change selection pressures. Antibodies from the female are passed to the yolk in large quantities. Yolk transmitted antibodies are highly resistant to degradation (Fichtali et. al., 1993). During embryonic development germinal cells are move into the yolk sac (Eyal-Giladi, 1993). Egg yolk antibodies are transferred to the embryo. The yolk is protected from kidney excretions by the allantois (Parkhurst and Mountney, 1988). Therefore, electrophoretograms of egg yolk proteins would have spots common to both males and females; for common metabolic and developmental pathways. Unique spots would represent sex-specific proteins when egg yolk sampling was similar except for sex segregation. Some of the sex-specific genes are known to be transcribed during embryogenesis but the protein translations are unknown.
It is well known by those skilled in the art and the subject of hundreds of articles, how to separate a protein from a mixture of proteins, isolate the separated protein, use the isolated protein in a vaccine and use the antibodies for diagnostic immunoassays (Belote et. al, 1985; Bradford et. al., 1976; O'Farrel et. al., 1975; Oakley et. al., 1980; Towbin, 1979; Tsang and Grunder, 1983; Zijpp et. al., 1985; Gassmann et. al., 1990; U.S. Pat. No. 5,080,895; U.S. Pat. No. 5,021,244).